Our aim is to dissect the interactions of p21 (a downstream effector of the tumor suppressor protein p53) with its two target, cyclin-cdks and PCNA. In year 1 we have established conditions for quantitatively analyzing the inhibition of cyclin-cdk by p21. This will enable us to measure the exact contribution of the interaction between the cyclin-binding Cy motif of p21 with a docking site on the cyclin in the inhibition of kinase activity. The conditions obtained have already demonstrated qualitatively the importance of the Cy motif in kinase inhibition. Different methods of site directed mutagenesis are being tried to determine the optimal method by which to generate a library of mutants in the Cy motif of p21. In year 2 this library of mutants will enable us to quantitatively determine exactly what constitutes a functional Cy motif. In order to dissect the interaction of p21 with PCNA we now have mutant forms of PCNA that fail to interact with p21 or with the exonuclease Fen 1 without disrupting the trimeric structure of PCNA. This is the first step towards analyzing how the p21-PCNA interaction impinges on DNA replication and repair in cancer cells.

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